Red blood cell (RBC) reversible aggregation is one of the main processes determining the blood microcirculation, it affects many aspects of physiology. Therefore, it is important to assess the underlying mechanisms of the cells interaction. However, until now there is only poor understanding of the cells interaction mechanisms. Two hypothetic mechanisms are generally considered based on the “depletion layer” and the “cross-bridging” mechanisms that are believed to be mutually exclusive. For both of these models direct experimental evidences are still missing. In this work, we aimed for a better assessment of the cells interaction mechanisms by implementing new methods. We studied the cells interaction using optical tweezers coupled with microfluidics and fluorescence microscopy. Implementation of optical tweezers allows for the measurement of a pure interaction between the cells at sub-pN range while having a precise control over the measurement process. Briefly, we performed a measurement of the RBC interaction in different aggregation inducing media such as plasma, serum and model solutions of macromolecules (fibrinogen, dextran, albumin, gamma globulin). We will present the following data:

The interaction forces in different media – the cells interaction forces are found to be in the range of few pN and dependent on the concentration of macromolecules in solution. The main result is the two times difference between the forces inducing two RBCs aggregation and dissociation processes. Each of these forces exhibited a different dependence on the macromolecule concentration. Consequently, it means that generation of each of the forces should be governed by different interaction mechanisms that should work at the same time.

Fibrinogen and dextran were found to adsorb onto the membrane of a single red blood cell. It is an important finding, because up to now a convincing results regarding adsorption was not available, and considered to be subject to numerous potential artefacts (e.g., trapped fluid between cells). In contrary, our result is a direct proof of adsorption.

Optical tweezers measure pure interaction of the cells and can serve to control the results of routine measurements. It is crucial to have control measurements due to a discrepancy between the results obtained by different measurement techniques. We found that the cup-bob and cone-plate measurement systems are subject to artefacts as they show a non-existing temperature dependence (with ~1.5 fold change) in the range of 20-38°C.
The results of our study provide a new insight on the cells interaction mechanism. They provide direct experimental evidences proving that both the “depletion layer” and the “cross-bridging” interactions should take place in RBC aggregation. On the other hand, optical tweezers can provide a pure measure of RBC aggregation capable of explaining the discrepancy between different measurement techniques.